Screening And Identification CTSD In NPC Interaction Proteins

ObjectiveScreening and identification Cathepsin D(CTSD) interaction proteins innasopharyngeal carcinoma（NPC）cells, to understand the function of CTSD in NPCinvasion and metastasis.MethodsIn this study, we collected the high metastatic5-8F NPC proteins, and performedco-immunoprecipitation (co-ip) with gel electrophoresis to separate CTSD interactionproteins, then applied Liquid Chromatogram electrospray ionization quadrupoletime-of-flight mass spectrometry (LC-ESI-MS/MS) to identified proteins thatassociated with CTSD in NPC5-8Fcell. Bioinformatics methods such as Geneontology, function clustering, signaling pathways and protein-protein interactionnetwork analysis were used comprehensively analysised of the CTSD interactors. Atlast, we verified two of these CTSD interaction proteins by co-ip combined withwestern blot.Results1、Co-ip combined with mass spectrometry were applied and identified143CTSDinteraction proteins.2、Gene Functional Classification showed that70CTSD interaction proteins in143CTSD interactors were divided into12classes, the function of these proteins mainlyinvoles: transmembrane transport, cytoskeleton, oxidative phosphorylation, proteinsynthesis, cell apoptosis, signal transfer, oxidoreduction, molecular chaperone,glycometabolism, etc. The other73CTSD interaction proteins was not clustering,3、GO_BP analysis showed that the protein biological process of143CTSDinteraction proteins mainly involves the stress reaction, negative control, metabolism,transport, localization, positioning, transport, etc. GO_MF analysis showed that the molecular function of CTSD interaction proteins involves protein bind, catalyticactivity, purine nucleotide binding, nucleotide, cytoskeleton, oxidoreduction,molecular structure activity, etc. GO_CC analysis analysis showed that the CTSDinteraction proteins subcellular localization in membrane, organelle, vesicle, polymercomposites, etc.4、Biocarta and KEGG signaling pathways found that143CTSD interaction proteinsinvolves two Biocarta signaling pathways: Hypoxia inducing factor in cardiovascularsystem, telomere, telomerase, aging, and nine KEGG pathway: oxidativephosphorylation, pyruvate metabolism, glycolysis pathway, adhesive connection,antigen processing and presented, proplonic acid metabolism, ribosomes signaling,the phosphate pentose pathway.5、Protein interaction network showed that EGFR、 HSP90A and CTSD constituteinteractome, which provide new clues for the study of the mechanism of CTSD inNPC invasion and metastasis.6、We utilized co-ip combined with Western blot and found that EGFR and HSP90Ainteract with CTSD, which verified the reliability of the mass spectrometry results.Conclusion1、Co-ip combined with mass spectrometry were applied and identified143CTSDinteraction proteins.2、Bioinformatics analysis found that EGFR, HSP90A composed a group withCTSD, which may play an important role in NPC invasion and metastasis.3、Co-ip combined with Western blot analysis showed that EGFR and HSP90Ainteract with CTSD, which was consistent with mass spectrometry results and verifiedthe reliability of the mass spectrometry results.